Historically, the mushroom has attracted considerable attention as a health-oriented food not only in China and Japan but world-wide. However, during the last decade, the anti-tumor effects of various kinds of mushroom components have been noted as a result of recent research in this field occasioned by the development of new analytical techniques to study the pharmacologically active components from mushrooms .sup.1,.sup.2,.sup.3,.sup.4,.sup.5. FNT .sup.1. M. Torisu et al., Significant Prolongation Of Disease-Free Period Gained By Oral Polysaccharide K (PSK) Administration After Curative Surgical Operation Of Colorectal Cancer. Cancer, Imm. Imm., 32: 261-168, 1990. FNT .sup.2. J. Akiyama et al., Immunochemotherapy Of Transplanted KMT-17 Tumor In WKA Rats By Combination Of Cyclophosphamide And Immunostimulating Protein-Bound Polysaccharide Isolated From Basidiomycetes. Cancer Res. 37:3042, 1977. FNT .sup.3. T. Mizuno et al., Antitumor Activity And Some Properties Of Water-Soluble Polysaccharides From "Ilimematsutake", The Fruiting Body Of Agaricus Blazei Murill. Agric. Biol. Chem. 4:2889-2896, 1990. FNT .sup.4. T. Mizuno et al., Antitumoractive Polysaccharides Isolated From The Fruiting Body Of Hericium Erinaceum, As Edible And Medicinal Mushroom Called Yamabushitake Or Houtou. Biosci. Biotech. Biochem., 56: 347-348, 1992. FNT .sup.5. "H. Kawagishi et al., Isolation And Characterization Of A Lectin From Grifola Frondosa Fruiting Bodies. Bioch. et Biophy. Acta. 1034: 247-252, 1990";
The immunological status of a patient has been recently regarded as an important factor in the control of cancer, especially when there exists only a low tumor burden. This has given rise to the concept of the use of Biological Response Modifiers (BRM) in cancer therapy. The BRM is defined as "drugs that can regulate the relationship between the host and the tumor, leading to a biological response of therapeutic value." Therefore, there is a need extant in the area of treating patients to control cancer (especially where there exist only a low tumor burden) by finding materials capable of exhibiting immunomodulating actions as well as tumor inhibition when used clinically on various types of carcinoma.
In the area of mushrooms, the most potent strain examined was Coriolus versicolor in which PSK (polysaccharide Krestin) was extracted from Basidiomycetes and reported from Japan in 1965.sup.6,.sup.7 and PSP (polysaccharide peptide) from Cov-1 (Yun Zhi) reported from China in 1984.sup.8. Many experimental studies and clinical investigations of PSK.sup.1,.sup.9 and PSP.sup.10 relate to their anti-tumor effect and especially for their potential use in cancer immunotherapy. It was found that the anti-tumor effect of PSP was more potent than that of PSK.sup.11. In vitro experiments of PSP were reported to inhibit the proliferation of P388 leukemia cells and Ehrlich ascites cells; it also inhibited the proliferations of some human tumor cell lines including SCG-7901, SPC, and SLY (4). In vivo experiments showed that PSP inhibited the growth of murine sarcoma 180 in tumor bearing mice.sup.12. The immunopotentiating effect of PSP was also noted, and it was seen that PSP increased the thymus weight and the serum C3 and IgG content of tumor bearing mice.sup.13. Furthermore, PSP promoted lymphocyte proliferation and increased the production of IL-2 and interferon (INF).sup.14. A clinical study at the Shanghai Medical University involves 151 cases of various kinds of cancer patients who were treated with PSP, and found noticeable anti-cancer effects without toxicity to the body.sup.10. Since the PSP used in these studies were in crude extracts, further purifications of PSP are needed, as the mechanism of the anti-tumor effect of PSP was not clear. FNT .sup.6. Y. Nakono et al., Influence Of Protein Polysaccharide (PS-K) Isolated From Basidiomycetes On Delayed Hypersensitivity In Sarcoma-180 Bearing Mice, Proc. Japan. Cancer Assoc., 32: 282, 1973. FNT .sup.7. S. Tsukagoshi et al., Kretin (PSK) Cancer Treat. Rev., 11-131-155, 1984. FNT .sup.8. Q. Yang et al., Isolation Of The Polysaccharide Components Of PSP J. Shanghai Teach. Univ. (Natural Sciences Ed) 4:36, 1986. FNT .sup.9. Y. Nio et al., In Vitro Immunomodulating Effect Of Protein Bound Polysaccharide, PSK On Peripheral Blood, Regional Nodes, And Spleen Lymphocytes In Patients With Gastric Cancer. Cancer Imm. Imrn. 32: 335-341, 1991. FNT .sup.10. T. Liu et al., Clinical Implication Of PSP In Oncology In Recent Advances In Cancer, Published By Cancer Research Group, CUHK, 57-62, 1989. FNT .sup.11. X. Li et al., A Study Of Anti-Cancer Effects Of PSP And PSK On Human Tumor Cell Lines In Vitro. Acta Acad. Med., Shanghai, 14:23-24, 1987. FNT .sup.12. J. Zhou et al., The Anti-Tumor And Immunomodulating Activity of PSP In Mice, J. Shanghai Teach. Univ. (Natural Sciences Ed.) 3: 72, 1988. FNT .sup.13. X. Li et al., "Immunomodulating Actions Of PSP, In Recent Advances In Cancer, published by Cancer Research Group, CUHK, pp. 45-56, 1989. FNT .sup.14. X. Li et al., Immune Enhancement Of A Polysaccharides Peptides Isolated From Coriolus Versicolor, Acta. Pharm. Sinica, 11: 542-545, 1990.
Antitumor substances from RNase containing sources such as bovine semen, pancreas, human pancreas, various kinds of bacteria have been reported (Newton, 1993). The antitumor effect of such RNase was recently summarized in a review paper (Youle et al 1993). RNase from bovine semen and human pancreas was reported to have immunosuppressive effects. An antitumor effect with a RNase has not been found in mushroom although there were some reports concerning the antitumor and immunomodulating effects of various kinds of polysaccharides peptides from several mushroom (Hotta et al, Cho et al).
The structure and amino acids sequence of any peptide extracted from Coriolus versicolor and having the mentioned biological activity has not been described previously. Previously, it was not clear whether the active principle was even a peptide. Since the active principle(s) are not known, it is of interest to find out the exact nature and the structure of such active principle's from Coriolus versicolor with antitumor activities.
A major object of the present invention is to provide a method for isolating polypeptides from crude PSP by high performance liquid chromatography (HPLC) and capillary isoelectrophoresis-focusing (CIEF) in order to ascertain the mechanism of the anti-tumor affect of PSP.
Another object of the present invention is to provide polypeptides of Coriolus versicolor that provide more potent anti-tumor affects than that of the crude extraction of PSP in which the polysaccharide peptide has a molecular weight of about 100 Kd.
A yet further object of the present invention is to provide purified Coriolus versicolor polypeptides having-a potent cytotoxic affects on human tumor cell lines but little affects on normal cell lines.
A further object still of the present invention is to provide purified Coriolus versicolor polypeptides that possess immunopotentiating affects as they increase white blood cells with increases of T and B lymphocytes, IgG and immune organs's weights, and wherein no toxic side affects are induced when these purified Coriolus versicolor polypeptides are administered in therapeutic doses as anti-cancer drugs for clinical use.
Still another object of the present invention is to isolate the active compound from PCV and determine its chemical composition for synthesis.
These and other objects of the invention will become more apparent by reference to the materials and methods hereinafter set forth.